Monoclonal human natural antibodies

ABSTRACT

Disclosed herein are hydridoma cell lines producing monoclonal human natural IgM antibodies and methods of use thereof. The antibodies are the monoclonal equivalents of circulating human natural antibodies. Also disclosed herein are pharmaceutical formulations and methods for treating HIV-1 infected individuals using the monoclonal human natural antibodies.

[0001] This application is a divisional of copending U.S. patentapplication Ser. No. 09/462,116, filed Dec. 28, 1999 which is a 371 ofPCT/US98/25258 Nov. 4, 1998 which claims benefit of 60/066,464 Nov. 24,1997.

BACKGROUND OF THE INVENTION

[0002] The effector molecules of the immune system include a repertoireof circulating immunoglobulins non-attributable to exogenous antigenicinduction, variously referred to as “autoantibodies” or “naturalantibodies”. The existence of such antibodies has been long recognizedand their various proposed functions may be classed as “self-attack” or“self-benefit”. For the former, the specter of autoimmunity is raisedand the term “autoantibodies” is customarily applied. For the latter,the term “autoantibodies” is customarily applied. For the latter,designated “natural; antibodies”, support of homeostasis is implied.

[0003] U.S. patent application Ser. No. 08/271,210 filed Jul. 5, 1994,discloses a circulating natural human antibody immunoreactive with anarginine-rich epitope present on human protamine. U.S. Pat. No.5,606,026 issued Feb. 25, 1997, discloses that the arginine-rich epitopeis present in the Tat protein of HIV-1 and further discloses a secondcirculating human natural antibody immunoreactive with a differentepitope on the Tat protein of HIV-1. In addition, a third circulatinghuman natural antibody immunoreactive with a cryptic epitope present onhuman lactoferrin is disclosed therein.

[0004] It has been shown that all three of the above-mentionedcirculating human natural antibodies decrease after HIV infectionreaching minimal levels as the patient progresses to AIDS. Theseantibodies are found in all sera of normal humans of all ages, from cordblood to adult, which, by virtue of their ubiquitous occurrence, areidentified as natural antibodies.

[0005] Therefore, what is needed in the art are the monoclonalcounterparts of these circulating human natural antibodies for theirtherapeutic and diagnostic uses.

SUMMARY OF THE INVENTION

[0006] The present invention provides monoclonal forms of human naturalantibodies.

[0007] In one aspect, the present invention provides hybridoma cell lineRWL-1 (ATCC CRL 12431), a product of the fusion of Epstein Barr virus(EBV) transformed umbilical cord blood cells and HMMA, mouse: humanheteromyeloma cells.

[0008] In another aspect, the present invention provides monoclonalhuman IgM antibodies, produced by RWL-1 cells.

[0009] In yet another aspect, the present invention provides anotherhybridoma cell line, RWT-4 (ATCC CRL 12472), a product of the fusion ofEBV-transformed umbilical cord cells with SHM-D33 cells (ATCC CRL 1668),mouse: human heteromyeloma cells.

[0010] In yet another aspect, the present invention provides monoclonalhuman IgM antibodies produced by RWT-4 cells.

[0011] In a still further aspect, the present invention provideshybridoma cell line RWT-12 (ATCC CRL 12477), a product of the fusion ofEBV-transformed human umbilical cord cells and HMMA, mouse: humanheteromyeloma cells.

[0012] In a still further aspect, the present invention providesmonoclonal human IgM antibodies produced by RWT-12 cells.

[0013] In a still further aspect, the present invention provides amethod for treating a patient suffering from an infection caused byHIV-1 comprising administering to a patient in need of such treatment aneffective amount for treating said infection of a monoclonal antibodyselected from the group consisting of antibodies produced by RWT-4cells, RWT-12 cells, and mixtures thereof.

[0014] In a still further aspect, the present invention provides amethod for increasing CD4+T cells in a patient suffering from aninfection caused by HIV-1 comprising administering an amount forincreasing CD4+T cells of antibodies produced by hybridoma cells havingAccession Nos. ATCC CRL 12472 , ATCC CRL 12477 and mixtures thereof.

[0015] In a still further embodiment, the present invention provides apharmaceutical formulation comprising isolated human IgM monoclonalantibodies selected from the group consisting of antibodies produced byhybridoma cell lines having Accession Nos. ATCC CRL 12472, ATCC CRL12477, mixtures thereof and a pharmaceutical acceptable vehicle.

[0016] These and other aspects of the present invention will be apparentto those of ordinary skill in the art in light of the presentdescription, claims and drawings.

BRIEF DESCRIPTION OF THE DRAWINGS

[0017]FIG. 1 (A-C) is an SDS PAGE of cyanogen bromide [CNBr] cleavedlactoferrin (LF) and SP80.

[0018] A. Protein stain. 1 mol. wt. markers; 2 LF(M) ; 3 SP80-basic; 4SP80-acidic. All three proteins (2,3,4) show identical cleavagefractions 1-8.

[0019] B. Immunotransfer with serum of rabbit immunized with SP80(acidic and basic) showing multiplicity of reactive sites and homologyof reactivity of LF(M) and SP80.

[0020] C. Immunotransfer with normal human male serum showing reactivitysolely with fraction 7 of each of the 3 proteins.

[0021]FIG. 2(A-C) is an Tricine SDS Page.

[0022] A. Protein stain. 1 mol. wt. markers; 2 LF(M); 3 SP80 (acidic andbasic). Resolution of fraction 7 shows 2 distinct bands.

[0023] B. Immunotransfer with normal human male serum showing reactivityspecifically localized in fraction 7B.

[0024] C. Immunoreactivity with fraction 7B of a monoclonal antibody(Mab) IgM from a human B cell derived hybridoma.

[0025]FIG. 3(A and B) shows in situ immunoreactivity, displayed by FITClabeled anti-human IgM, of a component of human sperm heads with : (A)human serum; (B) Mab reactive with LF fraction 7B.

[0026]FIG. 4(A-C) shows reactivity, by ELISA, of serum (diluted 1:100)of each of 5 males, 5 females and the Mab with: (A) 10 μg/ml of thecomplement of sperm coat proteins released following induction of theacrosome reaction in a suspension of swim-up spermatozoa; (B) 10 μg/mlof purified fraction 7B LF(M); C. 10 μg/ml native (non-denatured) LF(M).The relative reactivities of A. and B. indicate that a serum antibodyand the Mab are reactive with a specific component, but not all, of thesperm coat complement. The lack of reactivity with native LF (C)verifies that the natural antibody of serum and the Mab are reactivewith a site of LF that is not revealed in its native state.

[0027]FIG. 5. Sequences of the 12 —amino acid peptides representing theTat protein of HIV-1. Peptides 1-7 (SEQ ID NOS: 1 to 7) and 9-12 (SEQ IDNOS: 9 to 12) represent 5 residue overlaps. Peptide 8 (SEQ ID NO: 8) wasincluded to provide another variant of arginine distribution in order toascertain the maximum reactivity of human sera attributable to thearginine-rich region of Tat. Maximum titer with the arginine-rich region(peptides 7,8,9, SEQ ID NOS: 7, 8, 9) was, in fact, displayed withpeptide 8 (SEQ ID NO: 8). Maximum titer with the cysteine-rich region(peptides 4,5, SEQ ID NOS: 4, 5) was displayed with peptide 4 (SEQ ID NO4).

[0028]FIG. 6 (A and B).

[0029] A. IgM

[0030] B. IgG Analysis of reactivity of two cohorts of 70 human sera,HIV+ and HIV− (normal), with Tat protein of HIV. The HIV+ cohort wasassembled from sera collected prior to 1994, therefore thecharacteristics are not attributable to the anti-HIV medications in usesince that time. Each assay plate included both HIV+ and HIV− specimensand a single normal serum (ST). The recorded titer for each serum (X)represents X/ST.

[0031] The titers are grouped in intervals of 10 with the number of seraof each cohort designated for each interval. The distributions of bothIgM and IgG titers for the HIV+ sera are skewed to the lower intervals,particularly those of the IgM.

[0032]FIG. 7(A and B). Distribution of titers of A.IgM and B.IgG,reactive with peptide 8 (SEQ ID NO: 8) (FIG. 5) in each of two cohortsof 70 human sera, HIV+ and HIV− (normal). The preponderance of low ,orno titers of IgM and, even more strikingly, of IgG in the HIV+ seraindicates that depletion of the natural antibody reactive with thearginine-rich sequence of Tat is a correlation of the pathoprogressionof HIV.

[0033]FIG. 8(A and B). The distribution of titers of A. IgM and B. IgGreactivity with peptide 4 (SEQ ID NO: 4) (FIG. 5) in two cohorts of 70human sera, HIV+ and HIV− (normal), is in accord with the general trendof lower titers, in HIV+ sera, of the total Tat-reactive antibodies, butless stringent than that demonstrated for the titers of the peptide 8(SEQ ID NO: 8) reactive antibodies (FIG. 11).

[0034]FIG. 9 (A and B).

[0035] A. CD4+T cell counts.

[0036] B. Titers of IgM reactivity with Tat protein, peptide 4 (SEQ IDNO:4) and peptide 8 (SEQ ID NO: 8) (FIG. 5) of serial specimens from anHIV+ male over a period of five years preceding his death with adiagnosis of AIDS. Each specimen for CD4+T cell count was obtained atthe same time as that for serum analysis. The correlation of drop inCD4+T cells with the decline of titers of the natural antibodies isparticularly marked with respect to the titer of peptide 8 (SEQ ID NO:8) reactive antibodies, supporting the proposition that the decline inthat natural antibody may allow the T cell apoptosis, attributed to Tat,to proceed.

[0037]FIG. 10 (A and B).

[0038] A. CD4+T cell count.

[0039] B. Titers of IgM antibodies reactive with Tat protein, peptide 4(SEQ ID NO: 4) and peptide 8 (SEQ ID NO: 8) (FIG. 5) in serial specimensof sera, collected over a period of 9 years, from an HIV+ male whoseduration of infection is estimated at over 11 years, but has displayedno HIV associated pathology and has had no anti-HIV medication. Eachspecimen for serum analysis was obtained at the same time as that forCD4+ cell count. The titers and pattern of maintenance of titers of thenatural antibodies are correlative with maintenance of the CD4+T cellcounts within the normal range.

[0040]FIG. 11 (A and B).

[0041] A. CD4+T cell count.

[0042] B. Titers of IgM reactive with Tat protein, peptide 4 (SEQ ID NO:4) and peptide 8 (SEQ ID NO: 8) (FIG. 5) of serial specimens from anHIV+ male. Following the report of specimen 4, in which decline of CD4+Tcell count was noted, anti-HIV therapy was initiated. The count inspecimen 5, taken after 6 months of therapy showed significant rise andthe titer of IgM reactive with peptide 8, (SEQ ID NO: 8) underwent anexceedingly high rise. The successive specimens then showed maintenanceof CD4 T cell counts and natural antibody titers, concomitant withgenerally good clinical status.

DETAILED DESCRIPTION OF THE INVENTION

[0043] All patents, patent applications and literature references citedherein are hereby incorporated by reference in their entirety.

[0044] In one preferred embodiment, the present invention provides amonoclonal form of a human, natural IgM antibody immunoreactive with acrytic epitope present on human lactoferrin. This antibody is producedby hybridoma RWL-1, deposited with the American Type Culture collection(Manassas, Va.) on Nov. 14, 1997 and received ATCC Accession No. ATCCCRL-12431. The hybridoma was produced by fusing an Epstein Barr virustransformed human umbilical cord cell with a mouse: human heteromyelomacell as described in Example 1 below. The hybridoma produces humanmonoclonal antibodies of the IgM isotype. The fact that the antibodyproducing cell (the human umbilical cord cell) is of neonate origin andthe antibody is of the IgM isotype (and therefore does not cross theplacenta) demonstrates that this is indeed a natural antibody.

[0045] The IgM antibody immunoreactive with lactoferrin is characterizedas a natural antibody identified since it has been shown to be presentin a large cohort of normal human sera, and for which no pathologic roleor association is apparent. The reactive site for this natural antibodyhas been shown previously (3) and confirmed here, to be present in theplasma membrane complex of the human sperm head. These studies, designedto establish the molecular identity of that reactive site, haveconfirmed that an approximately 72.6 kD protein present in seminalplasma (2), accurately determined here as 80 kD, is also present in theprotein coat of the sperm head and that 8 kD protein is homologous with,and in fact is, lactoferrin. It is shown herein that the noted naturalantibody is specifically reactive with LF in a configuration other thanthat of the LF ubiquitous in body fluids. That configuration and thenatural antibody reactivity is revealed, in vitro (FIGS. 2, 4),following denaturation of native circulating LF, and is revealed, invivo (FIG. 3) in the LF incorporated in the protein coat of the humansperm head. LF is present in seminal plasma in the native configurationand, by a mechanism not yet determined, the antibody recognition form isassumed when it is deposited in the spermatozoal membrane/coat complex.The transition to that form and deposition in the sperm surface coatpresumably take place during the period of spermatogenic maturation inthe seminiferous tubules of the testes. It is relevant, therefore, tonote that large molecules such as immunoglobulins, particularly IgM, areexcluded from the lumina of the seminiferous tubules (24) and,therefore, from immunoreactivity with sperm components duringspermiogenesis. That barrier, however, does not exist in the femalereproductive tract, where the full complement of circulating antibodiesis present (23). Therefore, the LF reactive natural antibody isavailable for immunoreactivity with the LF of the sperm coat, followingejaculation into the female reproductive tract. That interaction maytake place in the sperm coat in situ as shown (FIG. 3) and is definitelycapable of taking place with the LF released, along with other coat andplasma membrane components (FIG. 4) as the sperm undergoes the sequenceof capacitation and acrosome reaction, which facilitate passage of thesperm through the protective zona pellucida surrounding the oocyte, andsubsequent entry into the oocyte (9, 10). Since the acrosome reactioninvolves fusion of the acrosomal membrane with the plasma membrane, thecomponents of the overlying protein coat are dispersed. Thus, thereleased LF could have ready access to the ooplasm were it not for thepresence, in the fertilization milieu, of the natural antibody capableof immunological nullification of the ability of that LF to endocytosethrough the oocyte membrane and, subsequently, to interact with the DNAof the gametes or pronuclei.

[0046] Among the many functions and interactions defined for LF, itscapacity to be endocytosed and interact with DNA is of increasinginterest ( 7, 8, 27-29). Particularly interesting are the recent reportsthat the interaction of LF with DNA is marked by sequence specificity(7). The underlying molecular bases for that specificity have not beendefined, but it is reasonable to expect that if LF/DNA interactionoccurs, in vivo it does so within a defined control system. It islogical, also, to propose that such a system exists in the organizedchromosomal complement of somatic cells, but not in the nascentundifferentiated complements of the pronuclei. Thus, in that context,the postulated control of sequence specificity in interaction of LF withDNA may not be operative. The presence of a natural antibody selectivelyreactive with LF in the specific configuration in which it exists in thesperm coat, but not with LF in its ubiquitous circulating form, mayrepresent a fortuitous natural selection mechanism on two bases: (1)inhibition of LF interaction with the DNA complements of the fertilizedoocyte and (2) restriction of immunoreaction by the circulating naturalantibody with LF at other loci, in its more prevalent, importantfunction-serving forms. The innate occurrence of that natural antibodyis verified since the hybridoma secreting the Mab, utilized to providesignificant data of this study, was derived from a human cord blood Bcell.

[0047] As shown below in Example 2, the antibody is immunoreactive withan epitope present on human lactoferrin, specifically the form oflactoferrin present in the protein coat of the human sperm head.Lactoferrin is an 80 kD glycoprotein present in the sperm head.Following induction of the acrosome reaction occurring duringfertilization, lactoferrin (which has been shown to interact with andbind to DNA) could potentially interfere with the interaction of spermand oocyte DNA. Therefore, one of the uses of the antibody of thepresent invention is as an additive to in vitro fertilization reactionsin order to prevent lactoferrin from interacting with sperm DNA prior tofertilization.

[0048] In alternative, preferred embodiments of the present invention,hybridoma cells producing monoclonal IgM antibodies immunoreactive withthe Tat protein of HIV-1 are provided. The hybridoma cell lines RWT-4and RWT-12 are immunoreactive with peptide 4 (SEQ ID NO: 4) and peptide8 (SEQ ID NO: 8), respectively, of FIG. 5. These hybridoma cells, as isthe case with hybridoma RWL-1, were produced by fusing EBV-transformedhuman umbilical cord cells with mouse:human heteromyeloma cells. RWT-4cells were deposited with the ATCC on Feb. 12, 1998 and receivedAccession No. ATCC CRL 12472 and RWT-12 cells were deposited on Feb. 25,1998 with the ATCC and received Accession No. ATCC CRL 12477. Theepitope specificity of each antibody is shown below in Example 3.

[0049] The monoclonal IgM antibodies produced by hybridomas RWL-1 (ATCCCRL 12341) RWT-4 (ATCC CRL 12472) and RWT-12 (ATCC CRL 12477) can beisolated from cultures of the cells that produce them and purified usingtechniques known to those of ordinary skill in the art, such as ammoniumsulfate precipitation, HPLC, column chromatography, etc.

[0050] The antibodies of the present invention are the monoclonalequivalents of the circulating IgM antibody identified in Science228:1211, 1985 (for RWL-1 cells) and described in U.S. Pat. No.5,606,026 issued Feb. 20, 1997 (for RWT-4 and RWT-12 cells). Thesecirculating antibodies are deficient in HIV-infected individuals anddecrease as AIDS approaches. Therefore, the monoclonal antibodiesproduced by ATCC CRL 12341, ATCC CRL 12477 and ATCC CRL 12472 can beused as positive controls in assays for prognosing the onset of AIDS.

[0051] In another preferred embodiment of the invention, a method fortreating a patient suffering from an infection caused by HIV-1comprising administering an effective amount to treat HIV-1 of naturalhuman IgM antibodies selected from the group consisting of antibodiesproduced by RWT-4 cells, antibodies produced by RWT-12 cells andmixtures thereof. It is envisioned that replenishment of the naturalantibodies deficient in HIV-1-infected and AIDS patients will be ofclinical benefit to these individuals.

[0052] As shown below in Example 5, the Tat protein of HIV-1 does notstimulate the induction of antibodies in humans (see Table I of Example5). This observation coupled with the fact that long term survivors(LTS)/long term non-progressors (LTNP), patients who are HIV-1 positivebut who do not exhibit any symptoms of the disease or progress to AIDS,have normal levels of the circulating natural antibodies equivalent tothe IgM antibodies produced by the hybridomas of the present invention.This demonstrates the utility of administering the monoclonal antibodiesof the present invention as a therapeutic to treat the disease. Due tothe fact that the Tat protein of HIV-1 has such an important role inestablishing and maintaining infection, and that the protein does notappear to be immunogenic in humans, administration of the monoclonalantibodies of the present invention to infected individuals is one wayto introduce antibodies specifically directed against the Tat protein.

[0053] The data of FIGS. 9, 10 and 11 clearly establish acorrespondence, in HIV+ humans, between the CD4+T cell count and theserum titer of the two IgM natural antibodies reactive with the Tatprotein of HIV, specifically with the sequences of the proteinrepresented by peptide 4 (SEQ ID NO: 4) and peptide 8 (SEQ ID NO: 8)(FIG. 10). The correspondence is more sharply shown with the antibodyreactive with peptide 8 (SEQ ID NO: 8), representing the arginine-richsequence of Tat.

[0054] Each of FIGS. 9, 10 and 11 display a unique example of thatcorrespondence. FIG. 9 shows the clinical report of CD4+T cell count andthe antibody assay data of a series of corresponding serum specimensfrom an HIV+ male over a period of 5 years preceding his death with adiagnosis of AIDS. FIG. 10 displays the corresponding data of specimensfrom an HIV+ male whose duration of infection is estimated at over 11years, but who has displayed no HIV-associated pathology and has had noanti-HIV medication. FIG. 11 displays the data of specimens from an HIV+male showing that, following institution of anti-HIV medication, bothCD4+T cell count and titers of Tat reactive antibodies, particularly theantibody reactive with peptide 8, rose to levels within the normalrange.

[0055] Since various Intravenous IgG (IVIG) preparations currentlycommercially available (e.g., from Sandoz Pharmaceuticals or CutterBiological) have been tested and certified for parenteraladministration, an IVIG preparation may be used as a vehicle foradministration of the monoclonal IgM antibodies of the presentinvention. These preparations have been shown to be safe for humanparenteral administration.

[0056] Generally, the dosage administered will, of course, varydepending upon known factors such as age, health and weight of therecipient, type of concurrent treatment, frequency of treatment, etc.Usually, a dosage of active ingredient can be between about 0.001 andabout 10 milligrams per kilogram of body weight. Precise dosage,frequency of administration and time span of treatment should bemonitored, for each individual, by determination of rise in CD4+T cellcount and other clinical indicia of relief from pathogeneticprogression.

[0057] In yet another preferred embodiment, a method for increasingCD4+T cell counts in a patient is provided comprising administering to apatient in need of such treatment, an effective amount to increase CD4+Tcell counts of antibodies selected from the group consisting ofantibodies produced by hybridoma ATCC CRL 12472, ATCC CRL 12477 andmixtures thereof. The effective amounts are the same as mentioned above.

[0058] For parenteral administration, the antibodies of the presentinvention can be formulated into pharmaceutical formulations or dosageforms as a solution, suspension, emulsion, or lyophilied powder inassociation with a pharmaceutically acceptable parenteral vehicle.Examples of such vehicles are water, saline, Ringer's solution, dextroseand 5% human serum albumin. In addition, as mentioned above, IVIGcommercially available preparations can be used as vehicles for deliveryof the antibodies of the present invention.

[0059] The pharmaceutical formulations of the present inventions do notneed to constitute an effective amount of the antibodies of the presentinventions since such amounts can be achieved by administering aplurality of such formulations.

[0060] The present invention is further described below in specificexample which are intended to further describe the invention withoutlimiting its scope.

EXAMPLE 1

[0061] The hybridoma, RWL-1, which secretes the monoclonal human IgMantibody reactive with a defined cryptic sequence of human lactoferrin,was created by fusion of a human umbilical cord blood B cell with thecell line HMMA, a human/mouse heteromyeloma (Posner M R, Ellorim H,Santos D. (1987) Hybridoma 6:611.) as set forth below.

[0062] The cord blood was obtained, at caesarian section, from a normal(but otherwise non-identified) neonate and the mononuclear cells wereisolated by density gradient centrifugation using FICOLL-PAQUE® (available from Pharmacia of Peapack, N.J.). The collected cells werewashed with RPMI-1640 medium (Sigma) and added to a culture ofEpstein-Barr Virus (EBV), (obtained from cultures of ATCC CRL 1612cells) in RPMI 1640 medium and incubated (37° C.) for 2 hours. The cellswere then spun down, resuspended in RPMI 1640, supplemented with fetalcalf serum (FCS), cyclosporin A, Pen/Strep (10 units Penicillin/100 mgStreptomycin per ml), and plated in 96 well plates at 30 cells/well.After 5 weeks incubation at 37° C. with periodic medium replenishment,the culture medium of each well was tested by ELISA (Pruslin F H,Winston R, Rodman T c. (1991) J. Immunol. Meth. 137:27) for the IgMmonoclonal antibody (Mab) specifically reactive with the 7B fraction ofdenatured lactoferrin (FIG. 2C).

[0063] Selected cultures of EBV immortalized B cells were grown to acell concentration of 10⁶ cells/well, then washed 5× in RPMI-1640(non-supplemented). The fusion partner (HMMA cells, described in Posner,M. R., et al., Hybridoma 6:, 611, 1987) were grown in RPMI 1640, FCS,Pen/Strep and azaguanine, and washed 3× in non-supplemented RPMI-1640°10⁶ cells were mixed with an equal number of the EBV immortalized cells.The mixed cell culture was spun, supernatant decanted and the cellsresuspended in warm (37° C.) 40% polyethylene glycol/RPMI-1640 (pH 7.2)and held for one minute. The cells were spun, washed 2× with RPMI, pH7.8, then resuspended (10⁶ cells/ml) in HY medium (Sigma), supplementedwith 20% FCS, HAT (Sigma), ouabain, Pen/Strep and plated out at 10⁵cells/well. After 3 weeks the growth positive wells were tested for thepresence of the specific antibody. The contents of the wells withpositive antibody were limited out (diluted) and replated at 0.5cells/well in HY/HT (Sigma), and supplemented with 20% FCS, SPIT(Sigma), Pen/Strep. The cells were grown for 5 weeks (37° C.) and thecontents of each well were retested for Mab specificity. Selectedcultures were incubated and grown to density of 10⁶ cells/ml. and spunat 400 RPM, 5 min. Each cell pellet was suspended in 5 ml 80% FCS, 10%DMSO and 10% RPMI-1640 and stored frozen at −70° C., in 2 ml aliquots.Stored aliquots have been defrosted and retested for viability andantibody specificity.

[0064] The hybridoma was deposited on Nov. 14, 1997 with the AmericanType Culture Collection (Rockville, Md.) and received ATCC Accession No.ATCC CRL 12431.

EXAMPLE 2

[0065] In the Example set forth below, the following Materials andMethods were used.

[0066] LF Proteins

[0067] Human milk lactoferrin, obtained from Sigma (L3770) is designatedLF (M). Seminal plasma LF was isolated from pooled specimens of semen,from clinically normal volunteer donors. Following liquefaction,sperm-free plasma was obtained by centrifugation and separated by DEAEion exchange chromatography (11) into a pool of basic and a pool ofacidic fractions. Each pool was subjected to gel filtration SEPHACRYL™ S300 HR, available from Amersham Pharmacia of Piscataway, N.J., and thefirst fraction of each pool was resolved at 80 kD and designatedSP80-basic and SP80-acidic, respectively.

[0068] Cyanogen Bromide (CNBr) Cleavage and SDS PAGE

[0069] CNBr treatment of SP80-basic, SP80-acidic and LF(M) was carriedout as described (12). Briefly, a 10 mg/ml 70% formic acid solution ofeach protein was incubated with CNBr (200 fold molar excess) at roomtemperature for 18-24 hr. Following lyophilization, the cleavagemixtures were electrophoresed on an SDS polyacrylamide gel (FIG. 1). Forenhanced resolution of the low molecular weight fractions (FIG. 2)electrophoresis was carried out on a 16.5% tricine gel (13).

[0070] Characterization of Fraction 7B

[0071] Fraction 7B was excised from the gel and extracted with H20-SDSwas precipitated by addition of KC1 and the component peptides of 7Bwere purified by dialysis against PBS (pH 7.2). Untreated sperm-freeseminal plasma proteins and native LF were PBS solutions. Determinationthat LF fraction 7B consisted of two peptides was carried out by theLaboratory of Mass Spectrometry at Rockefeller University, utilizingmatrix-associated laser desorption/ionization mass spectrometry (14).N-terminal sequencing of the peptides of LF fraction 7B was carried outby the Protein Sequencing Facility at Rockefeller University, utilizingrepeated cycles of Edman degradation followed by PTH analysis withmicrobore HPLC (15).

[0072] Immunoreactivity

[0073] Western blot was performed on Immobilon-P (Millipore) transfersof the electropherograms of LF(M) and acidic and basic SP80 andvisualized by chemiluminescence. ELISA was carried out by standardizedmethodology (16-18). Sera were those of a rabbit immunized with humanLF(M), a rabbit immunized with SP80 (acidic and basic combined) andhuman sera selected at random from a group of discards from clinicallaboratories, identified by gender, age and “no clinical findings”.Reactivity by all human sera was solely with fraction 7 of the PAGE(FIG. 1) and resolved at a distinct band designated 7B (FIG. 2).

[0074] Monoclonal Antibody (Mab) Specific for Fraction 7B

[0075] Mononuclear cells were isolated from cord blood of a normalneonate by density gradient centrifugation using FICOLL-PAQUE®(Pharmacia) and transformed with Epstein-Barr virus (19). Fusion withthe parental cell line HMMA, utilizing standard procedures (20),resulted in a set of IgM secreting hybridomas for which monoclonalitywas established by limiting dilution. Since reactivity of serum withdenatured milk LF(M) and SP80 was confined to a single PAGE fraction(FIG. 2) that fraction was isolated from the gel and utilized, togetherwith a set of proteins and peptides for which specific reactivity byother human natural antibodies has been established (16,18), as antigensin ELISA to screen those Mab's for exclusive reactivity with fraction7B.

[0076] Cytologic Localization of LF/SP 80 in Sperm Heads

[0077] A fraction of swim-up human sperm was obtained from spontaneouslyliquefied seminal plasma, washed 3 times with PBS, and finally suspendedin either human serum diluted 1:500 in PBS or in PBS solution of thepurified Mab, followed by overnight incubation at 40° C. Each suspensionwas washed 3 times with PBS and the collected sperm incubated in FITClabeled anti-human IgM (Sigma) for 1 hour. The sperm were washed withPBS, and a drop of the suspension placed on a slide, examined andphotographed, utilizing FITC-specific filters (FIG. 3).

[0078] Sperm Coat Protein Fraction

[0079] A fraction containing the components of the sperm coat wasobtained by induction of the acrosome reaction (21) in a suspension ofspermatozoa: the swim-up sperm were gently washed with PBS, collectedand suspended in Ca medium: 2 mM CaCl₂ 10 mM ionophore A23187(Calbiochem), 1 mM PMSF (Sigma) and incubated 4 hr at room temperature.The sperm cells were pelleted by low speed centrifugation and theresultant supernatant cleared of particles by high speed centrifugationfollowed by dialysis overnight at 40° C. The supernatant was tested byELISA, for reactivity with human sera and with the Mab reactive with LFfraction 7B (FIG. 4).

[0080] Results

[0081] The data reported here confirm previous studies indicating thatan 80 kD protein of human seminal plasma is homologous with LF (1,2).Fractionation of sperm-free seminal plasma by DEAE ion exchangechromatography (not shown) confirmed that the 80 kD protein is presentin two forms: basic and acidic, which contains the glycan moiety (22).CNBr cleavage fractions on SDS gels were identical for both forms ofSP80 as well as for LF derived from human milk (FIGS. 1A,2A). Also, thepattern of immunoreactivity of those fractions with serum of a rabbitimmunized with SP80 (FIG. 1) or with LF from human milk (not shown), arecorrespondingly identical. Similarly, prior reports (4) that normalhuman sera show no immunoreactivity with native LF from milk or withSP80 isolated from, or in the context of, seminal plasma are confirmed(FIG. 4). Especially significant is the confirmation (FIGS. 1, 2) that anatural antibody, identified in normal human sera (3,4), is reactivewith a cryptic sequence of LF and SP80 that is revealed upondenaturation of those proteins (FIGS. 1, 2). That sequence is segregatedin fraction 7B from the PAGE of CNBr cleavage products of LF(M) and SP80(FIG. 2). The innate occurrence of the natural antibody is strikinglydemonstrated by the derivation of a hybridoma from a cord blood cellwhich secretes an IgM/K that is specifically reactive with a componentof fraction 7B (FIGS. 2, 4).

[0082] Mass Spectrometry revealed that fraction 7B contains 2 peptides,10 kD and 9 kD. N-terminal sequencing identified DKVER (amino acidpositions 1-5 of SEQ ID NO: 13) for the load major peptide and SLDGG(amino acid positions 1-5 of SEQ ID NO: 27) for the 9 kD peptide. Uponthe assumption that CNBr cleavage of LF is at methionine residues and byreference to the published structure of LF (12) the sequence of each ofthe 2 peptides was derived and localized to the C lobe. A set of 12residue peptides, with 5 residue overlaps, comprising the derived linearsequences of the 2 peptides, was created (Table I). Thus far, specificreactivity of human serum IgM has not been identified with any one ofthose peptides tested singly, indicating that the fundamental epitopefor the natural antibody, although embodied in LF fraction 7B, isconformation dependent.

[0083] The localization of that epitope, in situ in the sperm head, isdemonstrated by cyto-immunoreactivity of human serum and by the Mabspecifically reactive with LF(M)/SP80 fraction 7B (FIG. 3) Furtherevidence that LF is present in the sperm coat proteins, in thatconfiguration in which the natural antibody epitope is revealed, isprovided by FIG. 4. Following induction of the acrosome reaction (21),resulting in dispersion of the protein coat/plasma membrane ensembleoverlying the acrosomal region of the sperm head, reactivity of acomponent of the coat with human serum IgM and with the Mab was shown(FIG. 4). Thus, FIGS. 3,4 provide evidence that, following the sequenceof capacitation and acrosome reaction, in vivo the LF shed from thesperm coat may be available for entry into the sperm-penetrated oocyte.However, since the complete immunoglobulin repertoire of plasma ispresent in the female reproductive tract (23) that availability may beinhibited by the natural antibody.

[0084] Table I. Overlapping duodecapeptides comprising the components ofLF fraction 7B. A. 10 kD, B. 9 kD A. Seq. ID No: B. Seq. ID No: D K V ER L K Q V L L H 13 S L D G G Y V Y T A C K 27 K Q V L L H Q Q A K F G 14V Y T A C K C G L V P V 28 Q Q A K F G R N G S D C 15 C G L V P V L A EN Y K 29 R N G S D C P D K F C L 16 L A E N Y K S Q Q S S D 30 P D K F CL F Q S E T K 17 S Q Q S S D P D P N C V 31 F Q S E T K N L L F N D 18 PD P N C V D R P V E G 32 N L L F N D N T E C L A 19 D R P V E G Y L A VA V 33 N T E C L A R L H G K T 20 Y L A V A V V R R S D T 34 R L H G K TT Y E K Y L 21 V R R S D T S L T W N S 35 T Y E K Y L G P Q Y V A 22 S LT W N S V K G K K S 36 G P Q Y V A G I T N L K 23 G I T N L K K C S T SP 24 K C S T S P L L E A C E 25 S P L L E A C E F L R K 26

[0085] As noted (Results) reactivity of human serum IgM or of the Mabwas not displayed against any of the peptides, indicating that theepitope is conformational.

[0086] References

[0087] 1. Hekman A, Rumke P. The antigens of human seminal plasma (withspecial reference to lactoferrin as a spermatozoa-coating antigen).Protides Biol Fluids 16:549-552, 1969.

[0088] 2. Goodman S A, Young L G. Immunological identification oflactoferrin as a shared antigen on radioiodinated sperm surface and inradioiodinated human seminal plasma. J. Reprod Immunol 21:99-108, 1981.

[0089] 3. Rodman T C, Laurence J, Pruslin F H, Chiorazzi N, Winston R.Naturally occurring antibodies reactive with sperm proteins: apparentdeficiency in AIDS sera. Science 228:1211-1215, 1985.

[0090] 4. Manchester K, Winston R., Rodman T C. Lactoferrin-reactivenatural antibodies. Ann NY Acad Sci in press.

[0091] 5. Boyden S V. Natural antibodies and the immune response. AdvImmunol 5:1-28, 1965.

[0092] 6. Guilbert B, Dighiero G, Avrameas S. Naturally occurringantibodies against nine common antigens in human serum. Detection,isolation and characterization. J Immunol 128:2779-1787, 1982.

[0093] 7. He J, Furmanski P. Sequence specificity and transcriptionalactivation in the binding of lactoferrin to DNA. Nature 373:721-724,1995.

[0094] 8. Bi B Y, Liu J L, Legrand D, Roche A -C, Capron M, Spik G,Mazurier J. Internalization of human lactoferrin by the Jurkat humanlymphoblastic T cell line. Eur J Cell Biol 69:288-296, 1996.

[0095] 9. Yanagimachi R. Mammalian fertilization. In: Knobil E, Neil JD, Eds. The Physiology of Reproduction. New York: Raven Press, p189-317,1994.

[0096] 10. Aitken R J. Fertilization and early embryogenesis. In HillierS G, Kitchener H C, Neilson J P, Eds. Scientific Essentials ofReproductive Medicine. London: W.B. Saunders, p2.10, 1996.

[0097] 11. Friesen A D, Bowman J M, Price H W. Column ion exchangepreparation and characterization of an Rh immune globulin forintravenous use. J Applied Biochem. 3:164-175, 1981.

[0098] 12. Metz-Boutigue M -H, Joll&s J, Mazurier J; Schoentgen F,Legrand D, Spik G, Montreuil J, Joll&s P. Human lactoferrin: amino acidsequence and structural comparisons with other transferrin. Eur JBiochem 145:659-676, 1984.

[0099] 13. Schagger H, van Jagow G. Tricine-sodium dodecylsulfate-polyacrilamide gel electrophoresis for the separation ofproteins in the range from 1 to 100 kDa. Anal Biochem. 166:368-373,1987.

[0100] 14. Beavis R C, Chait B T. High accuracy molecular massdetermination of proteins using matrix-assisted desorption massspectrophotometry. Anal Chem 62:1836-1840, 1990.

[0101] 15. Atherton D, Fernandez J, DeMott M, Andrews L, Mische S M.Routine protein sequence analysis below ten picomoles. In:Angeletti R H,Ed. Techniques in Protein Chemistry IV, Calif. Academic Press. p409-418,1993.

[0102] 16. Rodman T C, Pruslin F H, Chauhan Y, To S E, Winston R.Protamine-reactive natural antibodies in human sera. J Exp Med167:1228-1246, 1988.

[0103] 17. Pruslin F H, To S E, Winston R, Rodman T C. Caveats andsuggestions for the ELISA. J Immunol Meth 137:27-35, 1991.

[0104] 18. Rodman T C, To S E, Hashish H, Manchester K. Epitopes fornatural antibodies of human immunodeficiency virus (HIV)-negative andHIV-positive sera are coincident with two key functional sequences ofHIV Tat protein. Proc Natl Acad Sci USA 90:7719-7723, 1993.

[0105] 19. Chiorazzi N, Wasserman R L, Kunkel H G. Use of Epstein/Barrvirus transformed B-cell lines for the generation ofimmunoglobulin-producing human B cell hybridomas. J Exp Med 156:930-935,1982.

[0106] 20. Chiorazzi N, Generation of stable autoantibody-secreting Bcell hybridomas. Mol Biol Reports 16:65-73, 1992.

[0107] 21. Jamil K, White I G, Induction of acrosomal reaction in spermwith ionophore A23187 and calcium. Arch Androl 7:293-292, 1981.

[0108] 22. Spik G, Coddeville B, Mazurier J, Bourne Y, Cambillant C,Montreuil J. Primary and three-dimensional structure of lactotransferrin(lactoferrin) glycans. Adv Exp Med Biol 357:21-32, 1994.

[0109] 23. Yee A J, Silver L M. Contraceptive vaccine formulations withsperm proteins. In: Bronson R A, Alexander N J, Anderson D J, Branch DW, Kutteh W H, eds. Reproductive Immunology. Mass. Blackwell Science.part 2, chapt. 33, 1996.

[0110] 24. Haas G G Jr, Bronson R A, D'Cruz J, Fusi F M. Antispermantibodies and infertility In: Bronson R A, Alexander N J, Anderson, DJ, Branch D W, Kutteh W H, eds. Reproductive Immunology. Mass. BlackwellScience. part 2, Chapt. 7, 1996.

[0111] 25. Rodman T C, Pruslin F H, To S E, Winston R, Allfrey V G.Turnover of basic chromosomal, proteins in fertilized eggs: acytoimmunochemical study of events in vivo. J Cell Biol 90:351-361,1981.

[0112] 26. Monchev S, Tsanev S. Protamine-histone replacement and DNAreplication in the male mouse pronucleus. Mol Reprod Devel 25:72-76,1990.

[0113] 27. Fleet J C. A new role for lactoferrin: DNA binding andtranscription activation. Nutr Rev 53:226-231, 1995.

[0114] 28. Garre C, Bianchi-Scarra G, Sirito M, Musso M, Ravazzolo R.Lactoferrin binding sites and nuclear localization in K562 (s) cells. JCell Physiol 153:477-482, 1992.

[0115] 29. Hutchens T W, Henry J F, Yip T T, Hachey D L, Schanler R J,Motil K J, Garza C. Origin of intact lactoferrin and its DNA-bindingfragment found in the urine of milk-fed infants. Evaluation of stableisotopic enrichment. Ped Res 29:243-250, 1991.

[0116] 30. Concar D, The jaws of lactoferrin. Nature 344:710, 1990.

[0117] 31. Gerstein M, Andersen B F, Norris G E, Baker E N, Lesk A M,Clothia C. Two hinges produce a see-saw motion between alternativeclose-packed interfaces. J Mol Biol 234:357-372, 1993.

[0118] 32. Baker E N, Anderson B F, Baker H M, Day C L, Rumball S V,Smith C L, Thomas D H. Three dimensional structure of lactoferrin invarious functional states. Adv Exp Med Biol 357:1-12, 1994.

[0119] 33. Lonnerdal B, Iyer S, Lactoferrin: molecular structure andbiological function. Ann Rev Nutr 15:93-110, 1995.

EXAMPLE 3 Production of RWT-4 and RWT-12 Hybridoma Cell Lines

[0120] Each of the hybridomas was prepared by fusion of a humanumbilical cord blood B cell with a myeloma.

[0121] Myeloma fusion partner for RWT-4 was a heteromyeloma produced byfusion of a mouse and a human myeloma cell, obtained from ATCC: 5HMD33.

[0122] Myeloma fusion partner for RWT-12 was HMMA. (Posner, M.R. et al.,Hybridoma 6:611, 1987).

[0123] For each hybridoma, umbilical cord blood B cells were obtainedand immortalized as described above in Example 1.

[0124] After five weeks, the culture medium of each well was tested byELISA for reactivity with Tat protein. The media of selected cultures ofthose EBV-immortalized cells was then tested with each of the peptides(shown in Example 5, FIG. 5). Three of those displaying reactivity onlywith peptide 4 (SEQ ID NO: 4) (and at lower levels with peptide 5, SEQID NO: 5) and three displaying reactivity only with peptide 8 (SEQ IDNO: 8) (and at lower level with peptides 7, 9 (SEQ ID NO: 7, 9)) wereselected for fusion with the respective fusion partner as describedabove in Example 1. Note correction to that procedure: the fused cells(representing the hybridomas) were plated out at 0.5 cells per well toassure that no more than one cell was seeded into a well, thus assuringmonoclonality.

[0125] The cells were grown in NY/HT (Sigma), supplemented with 20%fetal calf serum, SPIT (Sigma) and Pen/Strep, to a density of 10⁶cells/ml, and spun at 400 RPM, 5 min.

[0126] Each pellet was suspended in medium, consisting of 80% fetal calfserum, 10% DMSO and 10% RPMI-1640, and stored, in 2 ml aliquots, at 70°C. or in liquid nitrogen. Those aliquots represent the hybridomas RWT-4and RWT-12 deposited with ATCC on Feb. 12, 1998 and Feb. 25, 1998,respectively.

EXAMPLE 4 Testing of Specific Reactivity of Hybridoma Cell Lines RWT-4and RWT-12

[0127] The culture medium of each, containing the specific IgMmonoclonal antibody secreted by the hybridoma, was treated for recoveryof the antibody.

[0128] The medium was concentrated in a CENTRICON® C-100 (available fromMillipore of Bedford, Mass.) column to remove salt and all proteins ofmol. wt. less than 100 Kd. The concentrated solution was then passedthrough a size exclusion gel on a Pharmacia S-300 column. The first peakof eluate was run on SDS polyacrylamide gel to inspect purity,demonstrated by display of two bands, representing the light and theheavy chain of the specific IgM, and no other bands. The eluate was thenreconcentrated in a new column to 200 μg/ml.

[0129] The light chain of each Mab was identified by ELISA withperoxidase labelled anti-gamma and anti-kappa.

[0130] The heavy chain (epitope specificity) of each was identified byELISA with the set of peptides. LIGHT CHAIN IDENTIFICATION ELISA : 1.unlabeled rb > IgM 2. Mab or total hu IgM (Sigma) 3. perox. labeledanti-kappa or anti-lamda Perox Labelled Anti-Lambda 0 ab Total IgM RWT-4Mab 1:4k  01 .24 .73 1:6k  01 .16 .54 1:10k 0 .12 .37 Anti-Kappa 1:4k 04 .55 .06 1:6k  01 .36 .04 1:10k 0 .23 .02 Anti-Lambda 0 ab Total IgMRWT-12 Mab 1:4k  02 .89 .03 1:6k  01 .65 .02 1:10k 07 .45 .01 Anti-Kappa1:4k  04 >1.00 .80 1:6k  03 .83 .57 1:10k 02 .56 .39

[0131] EPITOPE DETERMINATION IN TERMS OF TAT PEPTIDE SPECIFICITY STRWT-4 Mab RWT-12 Mab Pep 1 .01 .03 .05 2 .02 .02 .04 3 .03 .01 0 4 .48.94 .02 5 .20 .35 .12 6 .07 .01 .07 7 .16 .07 .36 8 .12 .07 .42 9 0 .02.01 10  0 .01 .03 11  0 0 0 12  .33 .16 .72 Total Tat Protein .49 .55.44

[0132] Peptide 12 (here) is designated peptide 8 in Example 5, FIG. 6.Therefore, here 7 7 12 8 8 9 9 10 10 11 11 12 12 8

[0133] These data represent the average, for each antibody/antigenreaction, of 20 separately run assays.

EXAMPLE 5

[0134] In the present Example, the following materials and methods wereused.

[0135] Sera

[0136] Human

[0137] The 70 HIV+ and 70 HIV− sera reported in FIG. 6 were collectedprior to 1994, and assayed for reactivity with Tat protein. Thereforethe characteristics of the HIV+ cohort are not attributable to theanti-HIV medications in use since that time. Of those 70 HIV+ sera, 52were available for the epitope analysis of Table I in which wereincluded 8 additional HIV+ sera for a total of 60 sera from HIV+individuals not on medication. The sera for the HIV+ serial sets (FIGS.9, 10, 11) were aliquots of specimens submitted for clinical examinationwith clinical data and concurrent medication noted. The 80 normal (HIV−)sera of Table I were assembled from specimens submitted forpre-employment examination identified only by age, gender and “noclinical findings”, and from donations by laboratory personnel.

[0138] Chimpanzees

[0139] A total of 22 sera from adult chimpanzees, certified as normal,were obtained: 16 (7 ♂, 9 ♀) from YERKES Regional Primate Center (EmoryUniversity); 6 (2 ♀, 4 ♂) from LEMSIP (NYU Medical Center). Serum of 1 ♂and 1 ♀ of the latter group were collected 22 months and 10 months,respectively, post innoculation with HIV infected cells.

[0140] Monkeys

[0141] A total of 32 sera from normal monkeys were obtained: 20 rhesusmacaques from YERKES, 1 from LEMSIP and 2 from LARC (Laboratory AnimalResearch Center, Rockefeller University), 4 pig tail macaques and 5baboons from LARC. Also, serum was obtained from 1 of the rhesusmacaques following innoculation with SIV (Mac 239) infected cells and 2specimens of rhesus plasma, 6 months post-innoculation with cell freesupernatant of SIV Mac 239 culture, were obtained from Dr. Lingi Zhong (Aaron Diamond AIDS Research Center, Rockefeller University).

[0142] Rabbits

[0143] Sera obtained from 20 (10 ♂, 10 ♀) New Zealand white rabbits(prior to any treatment) were generously provided by James Nolan(Hospital for Special Surgery, New York) and 10 were obtained from LARC.1 specimen of rabbit serum post-immunization with HIV Tat protein wasobtained from Intracel Corp (Isaquah, Wash.)

[0144] Mice

[0145] Sera from 30 normal adult mice: 12 Balb C, 6 C57 black, 2MRL-1pr, and 10 Swiss Webster were obtained through LARC. A series of 3immunizations with HIV Tat protein/adjuvant was administered to 1 Balb Cand 1 Swiss Webster and adjuvant alone was administered to 1 Balb C and1 Swiss Webster. Sera included in the data of Table I represent thespecimens collected 16 weeks after the final innoculation of each mouse.

[0146] Antigens

[0147] Recombinant Tat protein was obtained from Intracel Corp. inlyophylized form. Reactivity and working dilution for each vial of theprotein was standardized with a single (standard) human serum (16). Tatpeptides (SEQ ID NOS: 1 to 12) (FIG. 5), representing overlappingsequences in accordance with the published amino acid alignment of HIVTat (17) were prepared as previously described (14). The most recentreview (26) confirms that Tat is a highly conserved HIV protein withlittle digression from that sequence displayed by the various HIV lades.

[0148] Elisa

[0149] All sera were stored at −70° C. in small aliquots, to minimizethe effects of repeated freeze-thaw. The ELISA protocol has been rigidlystandardized and statistically evaluated (e.g. 15,16). Eachserum/antigen was tested in a minimum of 3 separate assays. Thecorrected serum O.D. for each antigen represented the read-out O.D. ofthe serum/antigen minus the O.D. of serum background (0 antigen).Corrected O.D. of 0.10 was considered positive. If corrected O.D. was0.08-0.15, the assay was repeated 3 additional times. For assay of humanand chimp sera, a single standard serum (ST) was included on each titerplate and the final titer was calculated as X/ST. Peroxidase labeledanti-human IgG or IgM (KPL) was used for all human and chimpanzee sera.Anti-monkey IgM or IgG (KPL) was found to be non-reactive withchimpanzee sera, but was appropriate by all criteria of specificity andserum-dilution proportionality with the different monkey sera tested.Similarly, the anti-mouse IgM or IgG (Sigma) and anti-rabbit IgM or IgG(KPL) were screened for specificity and dilution related gradient ofreactivity. Since the peroxidase labeled antibodies for each specieswere derived from goat serum, the ELISA included an extra blocking step,i.e. 1% normal goat serum applied following the antigen wash and priorto application of the species-specific test serum, to assure that nopart of the displayed reactivity was attributable to goat antibodies.TABLE 1 IgM IgG Species # of Sera Peptide: 1 4 8 Tat 1 4 8 Tat HumansMales 40 0 40 38 40 0 38 31 38 Females 40 0 40 40 40 1 40 36 40 HIV + 600 60 46 60 1 60 21 60 Chimps Males 11 0 11 10 11 0 11 9 11 Females 11 011 11 11 0 11 8 11 HIV + 2 0 2 2 2 0 2 2 2 Simmians Monkeys 32 0 0 0 0 032 2 21 SIV + 3 0 0 0 1 0 3 3 3 Rabbits Normal 30 0 2 0 0 0 3 0 0 Tat +1 1 0 0 1 1 1 0 1 Mice Normal 30 0 0 0 0 0 2 0 1 Adj. Only 2 0 0 0 0 0 00 0 Tat + 2 0 0 0 0 2 0 0 2

[0150] Results

[0151] Human

[0152]FIG. 6 presents the assay data of IgM and IgG reactivity with Tatprotein of HIV+ and HIV− (normal) sera. As noted in METHODS, those HIV+sera were collected from individuals who had not received any anti-HIVmedication other than that in general use prior to mid-1994 (e.g. AZT).Comparison of the assembly of titers of the two cohorts of 70 sera each,shows that the IgM titers (FIG. 6A) of the HIV+ cohort are atsignificantly lower levels than those of the HIV− cohort. Thedistribution of the Tat-reactive IgG titers of the same sera (FIG. 6B),however, appears to be random, both with respect to comparison of thetwo cohorts and, in individual sera (not shown), in relation to theTat-reactive IgM titers. Those IgG titers may represent maturation formsof the natural antibodies (27,28) or antibodies independently induced byunrelated antigens with sequences sufficiently concordant with regionsof Tat protein to be reflected as Tat-reactive.

[0153] Epitope analysis (Table I) of sera of each of the two humancohorts shows that the entire IgM reactivity with Tat protein is limitedto two non-adjacent sequences: one including peptides 4,5 embracing thecysteine-rich region and the other peptides 7,8,9 representing thearginine rich region (FIG. 1). In accord with the data of Table I, all(80) HIV− (normal) males and females have significant titers of IgMreactive with Tat protein as well as with the epitope represented bypeptide 4 (SEQ ID NO: 4), while all but 2 have significant titers withthat represented by peptide 8 (SEQ ID NO: 8). All of the 60 HIV+ serahave low, but significant, titers of IgM antibodies reactive with Tatprotein and the sequence represented by peptide 4 (SEQ ID NO: 4), whileonly 46 of the 60 have IgM reactive with the arginine-rich sequencerepresented by peptide 8 (SEQ ID NO: 8). For even those HIV+ sera thatare within the range of positive, the IgM reactivities with peptide 8(SEQ ID NO: 8) are at low levels (FIG. 7), clearly suggestive of a trendto depletion, more so than that of peptide 4 (SEQ ID NO: 4) (FIG. 8).Again, the IgG antibodies (Table I) may be considered to representmaturation forms (27, 28) of the IgM natural antibodies and/or thoseindependently induced by some exogenous antigenic factor. The latter isprobably applicable to the IgG reactive with peptide 1 (SEQ ID NO: 1)(FIG. 5), present in one HIV− serum, therefore not Tat induced, and oneHIV+ serum (Table I). The data of FIGS. 7 and 8 confirm that the declineof the Tat reactive natural antibodies is more stringently reflected inthat reactive with peptide 8 (SEQ ID NO: 8) (FIG. 7) than in thatreactive with peptide 4 (SEQ ID NO: 4) (FIG. 8). The correlation of thetiters of Tat reactive IgM natural antibodies with the pathoprogressionof HIV and with the CD4+T cell count, an established index of thatprogression (29), is shown in FIGS. 9, 10, 11. Each is a display of dataobtained from serial specimens of a single individual, including IgMassay titers for Tat protein, peptide 4 (SEQ ID NO: 4), peptide 8 (SEQID NO: 8) and clinical laboratory report of CD4+T cell counts. Theseries in FIG. 9 is that from an HIV + male collected over a period offive years preceding his death with a diagnosis of AIDS. Each value forTat protein IgM titer reflects the combination of the peptide 4 (SEQ IDNO: 4) and peptide 8 (SEQ ID NO: 8) IgM values for the same specimen.Particularly striking is the sharp rise followed by the precipitous dropin the peptide 8 (SEQ ID NO: 8) reactivity concurrent with the virtualwipe-out of the CD4+T cells in the specimen collected 8 months prior todeath. FIG. 10 is a display of data of the series of specimens from anHIV+ male whose duration of infection is estimated at over 11 years andwho has had no anti-HIV medication and no symptoms of HIV pathogenesisand, thus, fits the criteria of long-term -survivor (LTS) orlong-term-non-progressor (LTNP) (12,13). The pattern of maintenance oftiters of the IgM natural antibodies reactive with Tat protein, peptide4 (SEQ ID NO: 4) and peptide 8 (SEQ ID NO: 8) are similar to thosedefined for normal (HIV−) humans (14,16). The high levels of titers,particularly those for peptide 8 (SEQ ID NO: 8), are correlative withthe maintenance of CD4+T cell counts within the normal range. Similarcorrelation is shown in the series of specimens (FIG. 7) from a singleHIV+ individual for whom antiviral therapy was initiated followingreport of decline in CD4+T cell count. Following a period of medication,both CD4+T cell count and the titers of the natural antibodies,particularly those reactive with peptide 8 (SEQ ID NO: 8), rose. Thefollowing successive specimens showed maintenance of both CD4+T cellcounts and antibody titers in the normal range, concomitant with ageneral state of wellness of the patient.

[0154] Chimpanzee

[0155] The sera of all of the 22 normal chimps (Table I) had significanttiters of both IgM and IgG antibodies reactive with Tat protein andpeptide 4 (SEQ ID NO: 4). For peptide 8 (SEQ ID NO: 8), 21 of that groupdisplayed significant IgM and 17 displayed significant IgG reactivity.The sera of each of the 2 HIV innoculated chimps displayed significantIgM and IgG reactivity with Tat protein, with the sequences representedby peptides 4 (SEQ ID NO: 4) and 8, (SEQ ID NO: 8) and with no other.Thus, the natural antibody repertoire of chimpanzee is similar to thatof humans.

[0156] Monkey

[0157] No IgM reactive with Tat protein or any of its constituentpeptides was detected in the sera of any of the 32 normal sera (TableI). Of the 3 SIV infected monkeys, one showed reactivity with Tatprotein. All 32, however, displayed IgG reactivity with peptide 4 (SEQID NO: 4) and 21 of those displayed IgG reactivity with Tat protein. Twosera of the normal macaques and all three of the SIV infected macaquesdisplayed IgG reactivity with peptide 8 (SEQ ID NO: 8).

[0158] Rabbit

[0159] Of the 30 normal rabbit sera, two displayed IgM reactivity withpeptide 4 (SEQ ID NO: 4) which, however, was not accompanied bydetectable IgM reactivity with Tat protein. Those two and an additionalnormal rabbit serum displayed IgG reactivity with peptide 4 (SEQ ID NO:4) but, again, not with Tat protein. The Tat immunized rabbit serumdisplayed IgM reactivity with peptide 1 (SEQ ID NO: 1) and with Tatprotein and IgG reactivity with peptide 4 (SEQ ID NO: 4) as well aspeptide 1 (SEQ ID NO: 1) and Tat protein. That distribution suggeststhat the peptide 4 (SEQ ID NO: 4) IgM and IgG reactivity in both normaland Tat immunized rabbit serum reflects a response to an exogenousantigen that is not detectable in the assembled Tat protein. The IgM andIgG reactivity with peptide 1 (SEQ ID NO 1), displayed in the serum ofthe Tat immunized rabbit is attributable to induction by the immunogensince that peptide 1 (SEQ ID NO: 1) reactivity is reflected incomparably high reactivity with Tat protein.

[0160] Mouse

[0161] Of the sera from 30 normal, two Tat/adjuvant and twoadjuvant/only immunized mice, none displayed IgM reactivity with Tatprotein or any of the peptides (Table I). Two of the 30 normal mousesera displayed IgG reactivity with peptide 4 (SEQ ID NO: 4) and theserum of another mouse displayed IgG reactivity with Tat protein. Thesera of the two mice immunized with adjuvant/only displayed noreactivity while the sera of the two mice immunized with Tat/adjuvantdisplayed exceedingly high (>1.0) activity with peptide 1 (SEQ ID NO: 1)and with Tat protein. Clearly, for both rabbit and mouse, Tat protein isa potent inducer of an antibody response specifically directed to thesequence displayed in peptide 1 (SEQ ID NO: 1).

[0162] Discussion

[0163] The significance of the Tat protein is shown early in thepathogenetic sequence of HIV infection by its role in cell attachmentand entry of the virus. Evidence from in vitro study indicates that Tatparticipates in viral internalization, mediated primarily by the basicdomain (30,31), represented by Tat peptides 7, 8, 9 (SEQ ID NOS: 7, 8,9) (FIG. 5). Intracellular propagation of the virus is also dependentupon Tat through its interaction with the Tar region of the viral RNA,resulting in transactivation (18, 19). The cysteine region of Tat,represented by Tat peptides 4,5 (SEQ ID NOS 4, 5) (FIG. 5) plays anessential role in Tat/Tar binding and the consequent replication of HIV(18,19). Thus, two activities of Tat—mediation of viral cell entry andactivation of the internalized virus to replicate—are dependent upon thesequences of Tat that include the epitopes for the two natural IgMantibodies that are present in the sera of all human and chimpanzee seraexamined in this study, but are not present in the sera of othermammals, e.g. monkeys, rabbits, mice (Table I)

[0164] In accord with that epitopic specificity, we propose that thosenatural antibodies provide, or contribute to, the human host mechanismof resistance to HIV pathogenesis in the early post-HIV infectionperiod. Retardation of viral entry and intracellular replication bythose antibodies in the human host and absence of that retardation inrhesus macaques may account for the observations that T lymphocyteturnover in SIV infected rhesus macaques occurs at a considerably higherrate than that in HIV infected humans (32,33). Although the precisemechanisms whereby the CD4+T cell population is depleted in theperipheral blood cells of HIV+ humans are not yet specificallyestablished, a relationship between the CD4+T cell count and titers ofthe Tat-reactive natural antibodies is demonstrated in the serialspecimens of FIGS. 9, 10, 11 of this study. In each series, the CD4+Tcell counts parallel the maintenance and drop of the antibody titers.

[0165] However, the providential arrest of Tat-related pathogenicity bythose natural antibodies may be limited by the immune system recognitionof the antibody-reactive sequences of Tat as self antigens and theconsequent induction of tolerance (21, 22).

[0166] The separate and coordinate principles of innate and adaptiveimmunity have received much attention recently (6,7,8) which, hopefully,will provide further elucidation of the mechanisms and events of selfrecognition followed by tolerance. Thus far, the fundamental andimplemental event of self tolerance appears to be that of deletion, orturning off, of the T and/or B cells involved in natural antibodyproduction (23). Thus, as the Tat antigen load is increased, theproduction of Tat-reactive natural antibodies may be stifled,antibody-mediated restriction of the aggressive activities of Tat lost,and the period of pathoprogressive latency terminated. A pathogenicactivity of Tat, well documented in vitro, is that of induction ofapoptosis (29). The proposition that the Tat-reactive natural antibodiesmay impede the action of Tat, and thereby contribute to maintenance ofthe early period of apparent latency following HIV infection, issupported by the observation that persons designated LTS (long termsurvivor) (12) or LTNP (long term non progressor) (13) show littleevidence of T cell apoptosis (21) and, as we have shown (FIG. 10),maintain normal levels of the natural antibodies. In correlation arereports (34) that the resistance of chimpanzees to progress to AIDS isaccompanied by maintenance of T cell levels and little evidence of Tatinduced apoptosis.

[0167] Although the mechanisms underlying depletion of T cells byapoptosis are not completely understood, recent studies have establishedthat the Fas/Fas ligand system is not the modulating factor in HIVinduced apoptosis of CD4+T cells (35, 36). Particularly provocative,however, is a recent report that SIV (mac 239) induced apoptosis inperipheral blood mononuclear cells in vitro is mediated by the Fas/Fasligand system (37). That difference between SIV and HIV in the mechanismof apoptosis mediation is critically relevant to the thesis of thisstudy—that the interaction of HIV with the human immune system issignificantly unique. Another significant difference between HIV and SIVis indicated by in vitro studies of the effect of intervention byinterferon on viral replication. That effect appears to be primarilyconcerned with viral DNA synthesis which, in SIV infected cells, isblocked by interferon but, in HIV infected cells, is not (38).

[0168] Apoptosis of B cells as well as T cells has been attributed toaction by Tat (29). Even more compelling are the accumulating reports ofthe involvement of Tat in the neurodegeneration leading to dementia(24). The reports have included Tat dose-dependent apoptosis of humanfetal neurons in culture (39) and neuronal apoptosis detected in braintissue from patients who had died with a diagnosis of AIDS (40). Theprobability that the neurotoxic effects of Tat demonstrated in vitro mayoccur in vivo is supported by the potential ability of Tat to permeatethe blood brain barrier. Various analyses of the capacity for vascularpermeability and blood brain barrier passage by the sperm chromosomalprotein, protamine, have assigned that function to the arginineconcentration of protamine (41). That same capacity is inherent in thearginine rich sequence of Tat, represented here by peptides 7,8,9 (SEQID NOS: 7, 8, 9) (FIG. 5). Of particular relevance is the epitopeanalysis for the human natural antibody reactive with Tat peptide 8 (SEQID NO: 8) which showed (16) that the epitope for that natural antibodyis present in certain arginine-rich sequences of protamine as well as inHIV Tat protein.

[0169] The epitope similarity for the IgM and IgG for each of the twohuman natural antibodies suggests that each represents a pair of isotypeof the same antibody. We have previously proposed that the constancy ofIgM titers, but not the IgG titers, of the two natural antibodies inserial specimens, from each of a group of normal individuals, indicatesthat the IgM is the homeostasis—maintaining isotype (16) The mechanismand utility of class switch of natural antibodies are currently not wellunderstood nor readily apparent but, hopefully, will be clarified in thecourse of current investigations of the molecular and functional aspectsof the switch of isotype in innate as well as adaptive immunity (42).

[0170] Therefore, at present, assignment of separate roles to the IgMand IgG isotope of the human Tat reactive natural antibodies is notfeasible. However, it is clear that, in HIV+ humans, Tat-reactiveantibodies attributable to immunogenic induction do not occur (Table I).Since Tat-reactive antibodies are induced in monkeys, rabbits and mice(Table I), it appears that the failure is unique to the human immunesystem. Since chimpanzees are presumed to have high level of geneticidentity with humans (20), attribution of that uniqueness to geneticspecificity is supported by the profile of Tat reactive antibodies inthe chimpanzee sera (Table I). The parallel with human sera is evident:pre and post HIV infected chimps have IgG and IgM antibodies reactivewith Tat peptides 4 and 8 (SEQ ID NOS: 4 and 8) (FIG. 5) and with noother (Table I). However, the apparently greater innate resistance ofchimps to the pathoprogression to AIDS than that of HIV+ humans (10, 11)may be a departure from the genetic identity, possibly in some immunesystem component participating in induction of tolerance (21, 22). Thequestion then arises: is the same genetic characteristic related to theprotection against the ravages of HIV with which LTS/LTNP are endowed(12, 13)?

[0171] References for Example 5

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1 36 1 12 PRT Human 1 Met Glu Pro Val Asp Pro Arg Leu Glu Pro Trp Lys 15 10 2 12 PRT Human 2 Leu Glu Pro Trp Lys His Pro Gly Ser Gln Pro Lys 15 10 3 12 PRT Human 3 Gly Ser Gln Pro Lys Thr Ala Cys Thr Asn Cys Tyr 15 10 4 12 PRT Human 4 Cys Thr Asn Cys Tyr Cys Lys Lys Cys Cys Phe His 15 10 5 12 PRT Human 5 Lys Cys Cys Phe His Cys Gln Val Cys Phe Ile Thr 15 10 6 12 PRT Human 6 Val Cys Phe Ile Thr Cys Ala Leu Gly Ile Ser Tyr 15 10 7 12 PRT Human 7 Leu Gly Ile Ser Tyr Gly Arg Lys Lys Arg Arg Gln 15 10 8 12 PRT Human 8 Gly Arg Lys Lys Arg Arg Gln Arg Arg Arg Pro Pro 15 10 9 12 PRT Human 9 Lys Lys Arg Arg Gln Arg Pro Arg Arg Pro Gln Gly 15 10 10 12 PRT Human 10 Arg Pro Pro Gln Gly Ser Gln Thr His Gln Val Ser1 5 10 11 12 PRT Human 11 Thr His Gln Val Ser Leu Ser Lys Gln Pro ThrSer 1 5 10 12 12 PRT Human 12 Lys Gln Pro Thr Ser Gln Arg Gly Asp ProThr Glu 1 5 10 13 12 PRT Human 13 Asp Lys Val Glu Arg Leu Lys Gln ValLeu Leu His 1 5 10 14 12 PRT Human 14 Lys Gln Val Leu Leu His Gln GlnAla Lys Phe Gly 1 5 10 15 12 PRT Human 15 Gln Gln Ala Lys Phe Gly ArgAsn Gly Ser Asp Cys 1 5 10 16 12 PRT Human 16 Arg Asn Gly Ser Asp CysPro Asp Lys Phe Cys Leu 1 5 10 17 12 PRT Human 17 Pro Asp Lys Phe CysLeu Phe Gln Ser Glu Thr Lys 1 5 10 18 12 PRT Human 18 Phe Gln Ser GluThr Lys Asn Leu Leu Phe Asn Asp 1 5 10 19 12 PRT Human 19 Asn Leu LeuPhe Asn Asp Asn Thr Glu Cys Leu Ala 1 5 10 20 12 PRT Human 20 Asn ThrGlu Cys Leu Ala Arg Leu His Gly Lys Thr 1 5 10 21 12 PRT Human 21 ArgLeu His Gly Lys Thr Thr Tyr Glu Lys Tyr Leu 1 5 10 22 12 PRT Human 22Thr Tyr Glu Lys Tyr Leu Gly Pro Gln Tyr Val Ala 1 5 10 23 12 PRT Human23 Gly Pro Gln Tyr Val Ala Gly Ile Thr Asn Leu Lys 1 5 10 24 12 PRTHuman 24 Gly Ile Thr Asn Leu Lys Lys Cys Ser Thr Ser Pro 1 5 10 25 12PRT Human 25 Lys Cys Ser Thr Ser Pro Leu Leu Glu Ala Cys Glu 1 5 10 2612 PRT Human 26 Ser Pro Leu Leu Glu Ala Cys Glu Phe Leu Arg Lys 1 5 1027 12 PRT Human 27 Ser Leu Asp Gly Gly Tyr Val Tyr Thr Ala Cys Lys 1 510 28 12 PRT Human 28 Val Tyr Thr Ala Cys Lys Cys Gly Leu Val Pro Val 15 10 29 12 PRT Human 29 Cys Gly Leu Val Pro Val Leu Ala Glu Asn Tyr Lys1 5 10 30 12 PRT Human 30 Leu Ala Glu Asn Tyr Lys Ser Gln Gln Ser SerAsp 1 5 10 31 12 PRT Human 31 Ser Gln Gln Ser Ser Asp Pro Asp Pro AsnCys Val 1 5 10 32 12 PRT Human 32 Pro Asp Pro Asn Cys Val Asp Arg ProVal Glu Gly 1 5 10 33 12 PRT Human 33 Asp Arg Pro Val Glu Gly Tyr LeuAla Val Ala Val 1 5 10 34 12 PRT Human 34 Tyr Leu Ala Val Ala Val ValArg Arg Ser Asp Thr 1 5 10 35 12 PRT Human 35 Val Arg Arg Ser Asp ThrSer Leu Thr Trp Asn Ser 1 5 10 36 12 PRT Human 36 Ser Leu Thr Trp AsnSer Val Lys Gly Lys Lys Ser 1 5 10

What is claimed is:
 1. A hybridoma cell line having Accession No. ATCCCRL
 12472. 2. An isolated human IgM antibody produced by the hybridomaof claim
 1. 3. The hybridoma cell of claim 1 wherein said hybridoma isproduced by fusing Epstein Barr virus-transformed umbilical cord cellswith mouse: human heteromyeloma cells.
 4. The isolated antibody of claim2 wherein the light chain of said antibody is lambda.
 5. The isolatedantibody of claim 4 wherein said antibody is reactive with an epitopeconsisting of SEQ ID No.
 4. 6. The isolated antibody of claim 5 whereinsaid epitope is present in the Tat protein of HIV-1.
 7. The isolatedantibody of claim 6 wherein said antibody is a natural antibody.